The biochemistry of radicals, including carbon-based and protein radicals, has come under increasing scrutiny as radicals are progressively implicated in a wider and wider range of pathological conditions. Nevertheless, carbon radical biochemistry remains incompletely understood despite the value of carbon radicals as probes and their direct relevance to oxygen radical pathologies. The work proposed in this application builds on the following key advances made during the expiring period of support: (a) the demonstration that the prosthetic heme of lactoperoxidase, and probably all other mammalian peroxidases, is covalently bound to the protein by an autocatalytic process, (b) the unambiguous demonstration that lactoperoxidase protein radicals are formed during catalytic turnover of this enzyme, and (c) a growing understanding of the structural and catalytic roles of the active site residues in peroxidases. The first aim of the present project is to further characterize the structure of lactoperoxidase, with a focus on obtaining a crystal structure of the enzyme. The second aim is to elucidate the molecular level mechanism by which the prosthetic heme group is covalently bound to the protein, and the consequences of this covalent binding for catalysis. The third aim is to identify the set of amino acids on the surface of lactoperoxidase that are converted to radical centers during catalysis, and to elucidate their relationship to heme covalent binding and substrate oxidation. The fourth goal is to express catalytically active thyroid peroxidase in our baculovirus-insect cell system and to extend to it our investigation of the covalent binding of prosthetic heme groups and the formation and role of protein radicals in mammalian peroxidases. As part of these aims we propose to investigate the oxidation of substrates and inhibitors to radicals and the potential binding of these radicals to the lactoperoxidase and thyroid peroxidase protein radicals. The binding of both small molecules and macromolecules to the protein radicals may be related to pathologies associated with the reactions catalyzed by the mammalian peroxidases. The results should help to clarify the roles of peroxidatively generated carbon radicals in physiological and pathological processes and to provide insights into possible mechanisms for the modulation or suppression of such processes.